Sepsis remains one of the leading causes of death in burn patients who survive the initial insult of injury. Disruption of the intestinal epithelial barrier has been shown after burn injury; this can lead to the translocation of bacteria or their products (e.g., endotoxin) from the intestinal lumen to the circulation, thereby increasing the risk for sepsis in immunocompromised individuals. Since the maintenance of the epithelial barrier is largely dependent on the intestinal microbiota, we examined the diversity of the intestinal microbiome of severely burned patients and a controlled mouse model of burn injury. We show that burn injury induces a dramatic dysbiosis of the intestinal microbiome of both humans and mice and allows for similar overgrowths of Gram-negative aerobic bacteria. Furthermore, we show that the bacteria increasing in abundance have the potential to translocate to extra-intestinal sites. This study provides an insight into how the diversity of the intestinal microbiome changes after burn injury and some of the consequences these gut bacteria can have in the host.

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Background Inflammatory bowel disease (IBD) is established to drive pathological sequelae in organ systems outside the intestine, including the central nervous system (CNS). Many patients exhibit cognitive deficits, particularly during disease flare. The connection between colonic inflammation and neuroinflammation remains unclear and characterization of the neuroinflammatory phenotype in the brain during colitis is ill-defined. Methods Transgenic mice expressing a bioluminescent reporter of active caspase-1 were treated with 2% dextran sodium sulfate (DSS) for 7 days to induce acute colitis, and colonic, systemic and neuroinflammation were assessed. In some experiments, mice were prophylactically treated with paquinimod (ABR-215757) to inhibit S100A9 inflammatory signaling. As a positive control for peripheral-induced neuroinflammation, mice were injected with lipopolysaccharide (LPS). Colonic, systemic and brain inflammatory cytokines and chemokines were measured by cytokine bead array (CBA) and Proteome profiler mouse cytokine array. Bioluminescence was quantified in the brain and caspase activation was confirmed by immunoblot. Immune cell infiltration into the CNS was measured by flow cytometry, while light sheet microscopy was used to monitor changes in resident microglia localization in intact brains during DSS or LPS-induced neuroinflammation. RNA sequencing was performed to identify transcriptomic changes occurring in the CNS of DSS-treated mice. Expression of inflammatory biomarkers were quantified in the brain and serum by qRT-PCR, ELISA and WB. Results DSS-treated mice exhibited clinical hallmarks of colitis, including weight loss, colonic shortening and inflammation in the colon. We also detected a significant increase in inflammatory cytokines in the serum and brain, as well as caspase and microglia activation in the brain of mice with ongoing colitis. RNA sequencing of brains isolated from DSS-treated mice revealed differential expression of genes involved in the regulation of inflammatory responses. This inflammatory phenotype was similar to the signature detected in LPS-treated mice, albeit less robust and transient, as inflammatory gene expression returned to baseline following cessation of DSS. Pharmacological inhibition of S100A9, one of the transcripts identified by RNA sequencing, attenuated colitis severity and systemic and neuroinflammation. Conclusions Our findings suggest that local inflammation in the colon drives systemic inflammation and neuroinflammation, and this can be ameliorated by inhibition of the S100 alarmin, S100A9.

Intestine barrier disruption and bacterial translocation can contribute to sepsis and multiple organ failure- leading causes of mortality in burn-injured patients. Additionally, findings suggest ethanol (alcohol) intoxication at the time of injury worsens symptoms associated with burn injury. We have previously shown that interleukin-22 (IL-22) protects from intestinal leakiness and prevents overgrowth of Gram-negative bacteria following ethanol and burn injury, but how IL-22 mediates these effects has not been established. Here, utilizing a model of ethanol and burn injury, we show that the combined insult results in a significant loss of proliferating cells within small intestine crypts and increases Enterobacteriaceae copies, despite elevated levels of the anti-microbial peptide (AMPs) lipocalin-2. IL-22 administration restored numbers of proliferating cells within crypts, significantly increased Reg3β, Reg3γ, lipocalin-2 AMP transcript levels in intestine epithelial cells, and resulted in complete reduction of Enterobacteriaceae in the small intestine. Knockout of signal transducer and activator of transcription factor-3 (STAT3) in intestine epithelial cells resulted in complete loss of IL-22 protection, demonstrating STAT3 is required for intestine barrier protection following ethanol combined with injury. Together, these findings suggest IL-22/STAT3 signaling is critical to gut barrier integrity and targeting this pathway may be of beneficial clinical relevance following burn injury.

Over 1.4 million Americans have been diagnosed with inflammatory bowel disease (IBD), and ulcerative colitis (UC) makes up approximately half of those diagnoses. As a disease, UC cycles between periods of remission and flare, which is characterized by intense abdominal pain, increased weight loss, intestinal inflammation, rectal bleeding, and dehydration. Interestingly, a widespread recommendation to IBD patients for avoidance of a flare period is "Don't Drink Alcohol" as recent work correlated alcohol consumption with increased GI symptoms in patients with IBD. Alcohol alone not only induces a systemic pro-inflammatory response, but can also be directly harmful to gut barrier integrity. However, how alcohol could result in the exacerbation of UC in both patients and murine models of colitis has yet to be elucidated. Therefore, we conducted a retrospective analysis of patients admitted for IBD with a documented history of alcohol use in conjunction with a newly developed mouse model of binge alcohol consumption following dextran sulfate sodium (DSS)-induced colitis. We found that alcohol negatively impacts clinical outcomes of patients with IBD, specifically increased intestinal infections, antibiotic injections, abdomen CT scans, and large intestine biopsies. Furthermore, in our mouse model of binge alcohol consumption following an induced colitis flare, we found alcohol exacerbates weight loss, clinical scores, colonic shortening and inflammation, and propensity to infection. These findings highlight alcohol's ability to potentiate symptoms and susceptibility to infection in UC and suggest alcohol as an underlying factor in perpetuating symptoms of IBD.

Alcohol intoxication at the time of burn injury exacerbates post-burn pathogenesis. Recent findings suggest gut barrier integrity is compromised after combined alcohol and burn insult, which could contribute to these complications. Tight junction proteins and mucins play critical roles in keeping the gut barrier intact. Therefore, the goal of this study was to examine the effects of alcohol and burn injury on claudin and mucin expression in the intestines. We also evaluated if the combined insult differentially influences their expression in the small and large intestines. Male C57BL/6 mice were given a single dose of 2.9g/kg ethanol prior to a ~12.5% body area burn. One and three days following injury, we profiled expression of several tight junction proteins, mucin, and bacterial 16S rRNA genes in small and large intestine using qPCR. We observed >50% decrease in claudin-4 and claudin-8 genes in both ileal and colonic epithelial cells one day after injury. Claudin-2 was significantly upregulated, and occludin was down-regulated in small intestine one day following injury. Mucin-3 expression was substantially elevated (>50%) in small intestine, whereas mucin-2, and mucin-4 were considerably diminished in the colon (>50%) one day following injury. Most parameters were normalized to sham levels on day three, except for mucin-3 and claudin-8, which remained decreased in large intestine. Neither alcohol nor burn alone resulted in changes in junction or mucin gene expression compared to shams. This was accompanied with increases in the family of Gram-negative bacteria, Enterobacteriaceae, in both small and large intestine one day following injury. These findings suggest that alcohol and burn injury disrupts normal gut microbiota and alters tight junction and mucin expression in the small and large intestines.

Inflammasomes are multiprotein complexes that coordinate cellular inflammatory responses and mediate host defense. Following recognition of pathogens and danger signals, inflammasomes assemble and recruit and activate caspase-1, the cysteine protease that cleaves numerous downstream targets, including pro–IL-1β and pro–IL-18 into their biologically active form. In this study, we sought to develop a biosensor that would allow us to monitor the initiation, progression, and resolution of inflammation in living animals. To this end, we inserted a known caspase-1 target sequence into a circularly permuted luciferase construct that becomes bioluminescent upon protease cleavage. This biosensor was activated in response to various inflammatory stimuli in human monocytic cell lines and murine bone marrow–derived macrophages. Next, we generated C57BL/6 transgenic mice constitutively expressing the caspase-1 biosensor. We were able to monitor the spatiotemporal dynamics of caspase-1 activation and onset of inflammation in individual animals in the context of a systemic bacterial infection, colitis, and acute graft-versus-host disease. These data established a model whereby the development and progression of inflammatory responses can be monitored in the context of these and other mouse models of disease.

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